The present invention relates to the use of a sulfated polysaccharide with a molar mass of less than or equal to 10,000 g/mol, which can be obtained by radical depolymerization of a crude fucan derived from Phaeophyceae, for producing a medicinal product with activity against arterial thrombosis and against arterial restenosis.
Thrombosis consists of the formation of a clot (thrombus) in the circulatory system, this clot obstructing the lumen of the vessel in which it forms. It is the consequence of the pathological activation of the physiological phenomena of haemostasis, i.e. of the phenomena which contribute to the prevention and arrest of bleeding.
Thrombosis brings into a play a complex process involving the activation, in cascade, of various factors, resulting in the formation of thrombin, which is a key clotting enzyme, and then in fibrin formation. The formation of the thrombus begins with adhesion of the platelets to the subendothelial connected tissue, exposed by a lesion of the vascular endothelium. The platelets aggregate with one another and the aggregate becomes surrounded by a fibrin network which also traps white blood cells and red blood cells, forming the thrombus.
Arterial thrombosis differs from venous thrombosis in that, most commonly, it occurs on an artery in which there is a lesion due to the presence of an atheroma plaque; this lesion is also characterized by the proliferation and the migration toward the intima of the smooth muscle cells of the media. Arterial thrombosis often occurs when the atheroma plaque ruptures, with loss of continuity of the vascular endothelium. The adhesion and aggregation of platelets plays a primordial role in the phenomenon of arterial thrombosis.
The entire process of response to the lesion of an artery involves many cellular biological phenomena which involve modifications of the phenotype of smooth muscle cells (SMCs), and also the expression of growth factors which promote the proliferation of endothelial cells.
In the treatment of venous thrombosis, anticoagulants, in particular heparin, are conventionally used, which have the property of inhibiting thrombin and its formation.
Heparin is a sulfated polysaccharide consisting of units of glucosamine and of uronic acids which are 1,4-linked, in which the sulfate groups are present on the amine function of the glucosamine and/or on alcohol functions of the glucosamine and of the uronic acid. This polysaccharide, the anticoagulant properties of which are well known, is currently widely used in the treatment of thrombotic accidents. However, heparin has very significant side effects (bleeding, risk of immunoallergic thrombopenia) and it is relatively ineffective in arterial thrombosis. In addition, the animal origin of this product may cause a potential risk of contamination with unconventional infectious agents.
Techniques of chemical or enzymatic depolymerization have made it possible to produce, from NFH (nonfractionated heparin, the molecular weight of which is approximately 15,000 g/mol), polysaccharide chains of low molecular weight, i.e. of molecular weight of between 2000 and 10,000 g/mol, named LMWHs (low molecular weight heparins). Many LMWHs have been synthesized and are in particular marketed under the names Enoxaparin(copyright), Reviparin(copyright), Dalteparin(copyright), Fraxiparin(copyright), Tinzaparin(copyright), Certoparin(copyright), Opocrin(copyright), Parnaparin(copyright) etc.
Clinical studies have shown that the effectiveness of the LMWHs in the prophylaxis of venous thromboembolic accidents is identical to, if not greater than, that of NFH. However, the LMWHs do not abolish the hemorrhagic risk and can cause, just as NFH, although less frequently, immunoallergic thrombopenia.
Furthermore, it has been shown, in particular by M. Lerch et al. (European Heart Journal, August 1998, 19, 495) and H. Rickli et al. (European Heart Journal, August 1998, 19, 470), that LMWHs (Reviparin(copyright) and Fraxiparin(copyright) respectivelyxe2x80x99) are ineffective in combating restenosis after angioplasty, i.e. the phenomenon of reappearance of a stricture of the lumen of an artery linked to the involvement of a balloon catheter in vascular surgery.
Sulfated polysaccharides other than heparins exist, for example fucans. These sulfated polysaccharides, of high molecular weight (100 to 800 kDa), are present in the cell walls of the thalli of brown algae. They are polymers of sulfated L-fucose and may also contain D-xylose, D-galactose, D-mannose and uronic acids, the latter not being sulfated, contrary to those of heparin. Fucans also differ from heparin in that they do not comprise any amino sugars.
Fucans have various properties which make their use in many therapeutic domains particularly advantageous.
It has in particular been shown that fractions of low molar mass fucan, obtained by acid hydrolysis as described in European patent 0 403 377, have an anticoagulant (S. Colliec et al., Thromb. Res., 1991, 64, 143-154) and antithrombotic activity, when given intravenously (S. Mauray et al., Thrombosis and Haemostasis, 1995, 74(5), (280-1285) or subcutaneously (J. Millet et al., Thrombosis and Haemostasis, 1999, 81, 391-395) comparable to that of the low molecular weight heparins.
It has also been shown that these same fucan fractions are capable of inhibiting, like heparin, the growth of vascular smooth muscle cells in culture (D. Logeart et al., Eur. J. Cell. Biol., 1997, 74, 376-384 and 385-390). The effects observed are reversible, are not related to a cytotoxic action and depend on the concentration of the compound in the culture medium. This antiproliferative effect on the growth of smooth muscle cells appears to be specific since, at these concentrations, no inhibition is observed on the growth of fibroblast lines, and these compounds are observed to be capable of potentiating endothelial cell growth in culture (J. L. Giraux et al., Eur. J. Cell. Biol. 1998, 77, 352-359).
Giraux et al. have shown, in Thromb. Haemost., 1998, 80, 692-695, that the same fucan fractions, obtained by acid hydrolysis according to the protocol described in European patent 0 403 377, induce, in vitro, the release of TFPI (Tissue Factor Pathway Inhibitor) by human umbilical cord vein endothelial cells, this being an effect which may contribute to the antithrombotic action of these fucan fractions.
In addition, it has been shown, in Patent application EP 0 846 129, that fucan fractions, obtained by radical depolymerization of a fucan from Phaeophyceae in the presence of a metal catalyst and of hydrogen peroxide, and having a molar mass of less than or equal to 10,000 g/mol, conserve, in vitro, the anticoagulant properties of crude fucan. Such fucan fragments, obtained by radical depolymerization of a high molecular weight fucan, are different, with respect to their chemical structure, from fucan fragments obtained by acid hydrolysis of a crude fucan, as demonstrated in Patent application EP 0 846 129.
Besides NFH, LMWHs and fucans, other anticoagulants have been described for their antithrombotic action (inhibition of the formation of the thrombus and/or of its growth): these are in particular heparinoids (mixture of low molecular weight glycosaminoglycans, for example Orgarane marked by Organon Inc.), antivitamins K and hirudins. Hirudins, for example Lepirudin (Refludan(copyright)) marketed by Behrinwerke AGxe2x80x94Hoechst Marion Roussel or Desirudin (Revasc(copyright)) marketed by Novartisxe2x80x94Rhxc3x4ne Poulenc Rorer, can cause, in the same way as nonfractionated heparin, a considerable hemorrhagic risk.
Two classes of compounds can be used or are being studied in arterial thrombosis (Samama M. M. and Desnoyer P. C., xe2x80x9cLes bases pharmacologiques des traitements antithrombotiquesxe2x80x94Agents antithrombotiques actuels et futursxe2x80x9d [The pharmacological bases of antithrombotic treatmentsxe2x80x94Current and future antithrombotic agents], 1995, publisher Masson): there are anti-platelet aggregation agents, for example acetylsalicylic acid, dipyridamole, ticlopidine, clopidogrel and anti-GPIIb/IIIa antibody, and fibrinolytics (streptokinase, urokinase, etc.), which dissolve the clot by activation of the fibrinolytic system and release of plasmin (proteolytic enzyme capable of rapidly lysing the fibrin clot).
The use of these compounds must often be combined with an endoscopic intervention in order to widen the lumen of the narrowed artery, such as an angioplasty. Now, these treatments are not sufficiently effective in the medium term, since restenosis of the arteries in which occlusions have been removed in this way frequently occurs, even within a few months following the treatment.
Thus, it appears that the antithrombotic agents described above do not make it possible to effectively prevent or treat an arterial thrombosis or a restenosis, which is an essential problem after an angioplasty. Following such surgery, the endothelium exhibits dysfunction compared to the endothelium of origin. The parietal attack induced by this surgery may result in a phenomenon of decompartmentalization: loss of the endothelium and direct communication between the blood compartment and the smooth muscle cells. The wall responds to this attack via a process which combines migration, proliferation, destruction and secretion of extracellular matrix, via the smooth muscle cells positioned in the intima, followed by regrowth of the endothelium, which contributes to its thickening and therefore to the stricture of its lumen. This phenomenon of parietal cicatrization may contribute to the pathological condition itself, like anastomotic stenoses on vascular prostheses and restenoses after endoluminal dilation.
In view of the current state of the art, there is therefore a need for an agent with activity against arterial thrombosis and arterial restenosis.
The inventors have thus given themselves the aim of providing an agent which may be used for producing a medicinal product with activity against arterial thrombosis and arterial restenosis, said medicinal product:
having activity against vascular thrombosis, in particular against venous thrombosis, and particularly advantageously against arterial thrombosis, this being when it is administered parenterally,
not presenting any major hemorrhagic risk,
not having the potential risk of viral contamination related to products of animal origin,
making it possible to obtain prevention of arterial occlusion in the context of prevention of restenosis, in particular following an angioplasty.
The inventors have now shown that, unexpectedly, these aims are achieved by using a sulfated polysaccharide which can be obtained by radical depolymerization of a crude fucan derived from Phaeophyceae, said polysaccharide having a molar mass of less than or equal to 10,000 g/mol.
A subject of the present invention is the use of a sulfated polysaccharide which can be obtained by radical depolymerization of a crude fucan derived from Phaeophyceae, said polysaccharide having a molar mass of less than or equal to 10,000 g/mol, for producing a medicinal product intended to prevent or treat vascular thrombosis.
Particularly advantageously, the use of such a sulfated polysaccharide makes it possible to obtain an antithrombotic effect which is not accompanied by a major hemorrhagic risk.
Said sulfated polysaccharide can be obtained as described in Patent application EP 0 846 129. By way of example, the type of Phaeophyceae from which said sulfated polysaccharide is derived is Ascophyllum nodosum, Fucus vesiculosus, Pelvetia canaliculata or Undaria pinnatifida. 
Particularly advantageously, the plant origin of the polysaccharide used in the present invention eliminates any risk of viral contamination of the individual to which it is administered.
According to an advantageous embodiment of the use according to the invention, said medicinal product is intended to prevent or treat venous thrombosis.
According to another advantageous embodiment of the use according to the invention, said medicinal product is intended to prevent or treat arterial thrombosis, a process in which the formation of the thrombus and the platelet deposits play an important role.
According to an advantageous arrangement of this embodiment, said medicinal product is intended to prevent arterial restenosis, a phenomenon which is a precursor to arterial thrombosis.
Specifically, in the context of the prevention of arterial thrombosis, it is particularly advantageous to seek to prevent arterial restenosis, this being a pathological condition which, when it manifests itself, may result in a thrombosis of the artery.
The sulfated polysaccharide used in the present invention advantageously has a molar mass of less than 5000 g/mol.
According to another advantageous embodiment of the present invention, said medicinal product is intended to be administered parenterally, preferably intravenously or subcutaneously.
In rabbits, in an experimental model of venous thrombosis, after subcutaneous injection, the same antithrombotic activity is observed for doses of LMWH and of sulfated polysaccharide (polysaccharide which can be obtained by radical depolymerization of a crude fucan derived from Phaeophyceae, as described above) equal to 1 mg/kg and to 10 mg/kg, respectively, i.e. for a dose of sulfated polysaccharide 10 times greater than that of an LMWH.
If it is possible to extrapolate to humans, the daily doses of said medicinal product are preferably between 150 and 300 mg (preventive administration) or between 450 and 600 mg (curative administration) subcutaneously; it is clearly understood, however, that those skilled in the art will adjust the doses depending on the age, on the weight and on the pathological condition of the patient, in particular depending on the thrombogenic risk.
In the embodiment relating to the prevention of arterial restenosis, said medicinal product is advantageously intended to be administered locally, for example by endoparietal diffusion.
Endoparietal diffusion consists of local administration of the pharmaceutical composition, for example by means of a balloon. The repeated passage of the balloon, during the surgical intervention of angioplasty, has the effect of eliminating the endothelium and stressing the muscle cells and the extracellular matrix of the media. In response to the de-endothelialization, platelet aggregation will recur at the site of the lesion, locally releasing PDGF (Platelet Derived Growth Factor). In response to the medial stress, the intracellular FGF stored in the matrix will be released. These growth factors will induce the activation, migration and proliferation of the smooth muscle cells.
The technique of endoparietal diffusion allows endovascular topical distribution of the active principle present in said pharmaceutical composition. Various types of balloon may be used, such as catheters with dual balloons isolating an infusion chamber or gel-coated balloons, or nonoccluding catheters.
Besides the above arrangements, the invention also comprises other arrangements which will emerge from the following description, which refers to examples of measuring the antithrombotic activity, the hemorrhagic risk and the anticoagulant effect of sulfated polysaccharides obtained by depolymerization of a fucan from Phaeophyceae, and also to the study of the effects of these polysaccharides on platelet aggregation.
It should be clearly understood, however, that these examples are given only by way of illustration of the subject of the invention, of which they in no way constitute a limitation.